利用HPLC分析菠萝愈伤组织DNA甲基化水平  

Analysis of DNA Methylation Levels in Pineapple Callus by HPLC

作　　者：林文秋[1,2,3] 肖熙鸥 张红娜[1,2,3] 刘胜辉 孙伟生[1,2] 李运合 吴青松[1,2,3] LIN Wenqiu;XIAO Xi＇ou;ZHANG Hongna;LIU Shenghui;SUN Weisheng;LI Yunhe;WU Qingsong(South Subtropical Crop Research Institute,Chinese Academy of Tropical Agricultural Sciences,Zhanjiang,Guangdong 524091,China;Hainan Provincial Engineering Research Center for Pineapple Germplasm Innovation and Utilization,Zhanjiang,Guang-dong 524091,China;Key Laboratory of Tropical Fruit Biology,Ministry of Agriculture,Zhanjiang,Guangdong 524091,China)

机构地区：[1]中国热带农业科学院南亚热带作物研究所,广东湛江524091 [2]海南省菠萝种质创新与利用工程技术研究中心,广东湛江524091 [3]农业部热带果树生物学重点实验室,广东湛江524091

出　　处：《热带作物学报》2018年第11期2220-2224,共5页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金面上项目"菠萝组织培养过程中体细胞无性系变异的研究"(No.317244);海南省自然科学基金面上项目"不同菠萝品种对乙烯诱导成花敏感性差异的分子机理研究"(No.20163108);农业农村部物种品种资源保护(热带作物)项目"菠萝种质资源保护"(No.151821301354052716)

摘　　要：建立菠萝DNA甲基化水平的HPLC测定方法,分析菠萝愈伤组织DNA甲基化水平变化,为进一步研究菠萝体细胞无性系变异机理奠定基础。通过对流动相和水解温度等条件的优化,建立菠萝DNA甲基化水平的检测方法。结果表明,分离C和5m-C的最佳流动相为甲醇∶磷酸二氢钾∶三乙胺为10∶90∶0.2(V/V),pH 3.0,DNA的最佳水解温度为90℃。利用此体系分析菠萝愈伤组织和胚性愈伤组织的DNA甲基化变化,结果表明,菠萝愈伤组织在分化过程中DNA总甲基化水平呈动态变化,变化范围为5.14%～96.86%。此外,胚性愈伤组织甲基化水平低于非胚性愈伤组织。推测DNA甲基化影响菠萝愈伤组织的分化及胚性愈伤组织的形成。In order to lay a foundation of the mechanism of somaclonal variation in pineapple, the analysis of the DNA methylation levels of callus was established by HPLC. A method for the determination of the DNA methylation levels was established by optimized the mobile phase and hydrolysis temperature. The results showed that the optimum mobile phase was the combination A（10% V/V methanol, 90% V/V KH2 PO4, 0.2% V/V triethylamine in H3 PO4 adjusted to pH 3.0）. The hydrolysis temperature was 90 ℃. The analysis of the DNA methylation levels in pineapple callus indicated that the DNA methylation levels showed dynamic changes with pineapple callus which ranged from 5.14% to 96.86%. In addition, the DNA methylation level of embryogenic callus was lower than non-embryogenic callus. It is speculated that the differentiation of callus and formation of embryogenic callus were influenced by DNA methylation.

关 键 词：菠萝 愈伤组织 DNA甲基化 HPLC 

分 类 号：S668.3[农业科学—果树学]